The mammalian middle ear is unique among tetrapods (mammals, amphibians, reptiles, and birds), in that it contains three distinct ossicles (the malleus, incus, and stapes) that form an indirect and usually flexible coupling path between the eardrum and cochlea, for which the majority of the ossicular mass is concentrated away from the cochlear entry axis. This differs markedly from the middle ears of non-mammalian tetrapods, in which eardrum motions are transmitted to the cochlea more or less in a straight line via a rod-like columella structure. Many theories have been presented as to the functional consequences and possible advantages of this peculiar middle-ear arrangement, especially considering that mammals are capable of hearing to much higher frequencies than non-mammals (>100 kHz vs. <12 kHz), and these theories range from allowing the ossicles to adopt lower-inertia vibrational modes at higher frequencies; to providing added flexibility to protect th cochlea against high static pressures in the ear canal or impulsive stimuli; to leveraging the off-axis mass distribution and joint flexibility to reduce the amount of ossicular inertia transmitted o the cochlea in response to skull vibrations from self-generated sounds due to vocalizations, breathing, pumping blood, etc., and thus allowing more attention to be focused on the external sounds critical for survival. In this proposal, the functioning of the mammalian ossicular chain will be tested, using measurements of 3D motion due to air and bone-conducted stimuli and 3D displacements due to positive and negative static pressure in the ear canal; and modeled, producing anatomically accurate CT-based 3D models of the full middle ear with accurate representations of ossicular-joint flexibility and the optional incorporation of a coiled cochlear model for testing bone-conduction responses. The measurements will be performed, and models based, on human, cat, and mouse temporal bones, with their: 1) natural ossicular chains, as well as with modified versions that 2) alter its natural freedom of motion by fusing one or both ossicular joints, and that 3) remove its indirect coupling path and off-axis mass distribution by replacing the incus with a columella-like prosthesis directly connecting the long process of the malleus to the footplate or head of the stapes. The measurements and resulting models will be used to comparatively examine the role of middle-ear structure on: 1) sound transmission from the ear canal to the cochlea, especially at higher frequencies; 2) protection of the cochlea; and 3) the transmission of sound to the cochlea via bone conduction. Comparisons across the three proposed scientifically and clinically important mammalian species will help to clarify any functional changes caused by differences in mammalian middle-ear anatomy, while comparisons across the proposed ossicular chain modifications will allow clarification of the functional implications of the structural features unique to mammalian middle ears. The measurements and models with a prosthesis replacing the incus may additionally lead to new ideas for improving the clinical outcomes of middle-ear reconstruction surgeries.